As used herein, the term "ligand-receptor" assay refers to an assay for an analyte which may be detected by the formation of a complex between a ligand and another substance capable of specific interaction with that ligand, i.e., ligand receptor. The ligand may be the analyte itself or a substance which, if detected, can be used to infer the presence of the analyte in a sample. In the context of the present invention, the term "ligand", includes haptens, hormones, antigens, antibodies, deoxyribonucleic acid (DNA), ribonucleic acids (RNA), metabolites of the aforementioned materials and other substances of either natural or synthetic origin which may be of diagnostic interest and have a specific binding partner therefor, i.e. the ligand receptor of the ligand-receptor assay. In the context of the present invention the term "ligand receptor" includes materials for which there is a specific binding partner, i.e. the ligand of the ligand-receptor assay. Those skilled in the art will appreciate that the analyte of interest, a member of a specific binding pair may be either ligand receptor or ligand depending upon assay design.
Ligand-receptor assays are generally useful for the in vitro determination of the presence and concentration of ligands in body fluids, food products, animal fluids, and environmental samples. For example, the determination of specific hormones, proteins, therapeutic drugs, and toxic drugs in human blood or urine has significantly improved the medical diagnosis of the human condition. There is a continuing need for simple, rapid assays for the qualitative, semi-quantitative, and quantitative determination of such ligands in a sample. Furthermore, in many situations, such assays need to be simple enough to be performed and interpreted by non-technical users.
Ligand-receptor assays rely on the binding of ligands by receptors to determine the concentration of ligands in a sample. Ligand-receptor assays can be described as either competitive or non-competitive. Non-competitive assays generally utilize receptors in substantial excess over the concentration of ligand to be determined in the assay. Sandwich assays, in which the ligand is detected by binding to two receptors, one receptor labeled to permit detection and a second receptor frequently bound to a solid phase to facilitate separation from unbound reagents, such as unbound labeled first receptor, are examples of non-competitive assays. Competitive assays generally involve ligand from the sample, a ligand analoque labeled to permit detection, and the competition of these species for a limited number of binding sites provided by the ligand receptor. Those skilled in the art will appreciate that many variations of this basic competitive situation have been previously described and will not be discussed in detail herein except where pertinent to the general objectives of this invention. Examples of ligands which are commonly measured by competitive ligand-receptor assays include haptens, hormones and proteins. Antibodies that can bind these classes of ligands are frequently used in these assays as ligand receptors.
Competitive ligand-receptor assays can be further described as being either homogeneous or heterogeneous. In homogeneous assays all of the reactants participating in the competition are mixed together and the quantity of ligand is determined by its effect on the extent of binding between ligand receptor and labeled ligand analogue. The signal observed is modulated by the extent of this binding and can be related to the amount of ligand in the sample. U.S. Pat. No. 3,817,837 describes such a homogeneous, competitive immunoassay in which the labeled ligand analogue is a ligand-enzyme conjugate and the ligand receptor is an antibody capable of binding to either the ligand or the ligand analogue. The binding of the antibody to the ligand-enzyme conjugate decreases the activity of the enzyme relative to the activity observed when the enzyme is in the unbound state. Due to competition between unbound ligand and ligand-enzyme conjugate for antibody binding sites, as the ligand concentration increases the amount of unbound ligand-enzyme conjugate increases and thereby increases the observed signal. The product of the enzyme reaction may then be measured kinetically using a spectrophotometer.
In general, homogeneous assay systems require both an instrument to read the result and calibration of the observed signal by separate tests with samples containing known concentrations of ligand. The development of homogeneous assays has dominated competitive assay research and has resulted in several commercially available systems. Such systems are not, however, capable of providing results for the determination of multiple ligands in a sample in a single-test format not requiring instrumentation.
Heterogeneous, competitive ligand-receptor assays require a separation of bound labeled ligand or receptor from the free labeled ligand or receptor and a measurement of either the bound or the free fraction. Methods for performing such assays are described in U.S. Pat. Nos. 3,654,090, 4,298,685, and 4,506,009. Such methods, however, are not capable of providing semi-quantitative or quantitative results for the determination of ligands in a sample without using additional tests to calibrate the assay response.
The need for ligand-receptor assays that can be performed without the use of instrumentation has led to the development of immunoassays that are simple to perform and result in a response that can be visually interpreted. U.S. Pat. Nos. 4,125,372, 4,200,690, 4,246,339, 4,366,241, 4,446,232, 4,477,576, 4,496,654, 4,632,901, 4,727,019, and 4,740,468 describe devices and methods for ligand-receptor assays that develop colored responses for visual interpretation of the results. While such devices provide simple formats for the visual interpretation of assay results, only the presence or absence of ligand can be determined; semi-quantitative or quantitative determinations using these methods require that separate tests utilizing standards of known concentration be performed to establish the relationship between the observed response and the concentration of ligand.
Methods also have been developed for the internal calibration of ligand-receptor assays by providing devices that incorporate reference zones where the response at the reference zone represents the assay response for a particular concentration of ligand. The response generated by the unknown concentration of ligand in the sample at a test zone is compared with the response at the reference zone to determine the concentration of ligand in the sample either semi-quantitatively or quantitatively. European Patent Application No. 87302403.8 describes methods for using such internal references in noncompetitive sandwich assays to provide semi-quantitative determinations from visual reading of the results and quantitative determinations from instrumental reading of the results. Likewise, U.S. Pat. No. 4,540,659 and European Patent Application No. 85307785.7 describe systems incorporating references that provide the ability to make semi-quantitative determinations in competitive ligand-receptor assays that are visually interpreted. Both of these systems provide a visual interpretation of the amount of labeled ligand analogue bound to solid phase immobilized receptor.
Employing the techniques described for competitive ligand-receptor assays, the intensity of the resulting color is inversely related to the concentration of ligand in the sample such that assay results that are more intense in color than the reference are interpreted to mean that the sample contained ligand at a lower concentration than that represented by the concentration by the reference. A serious drawback, however, to the widespread utilization of such visually interpreted, competitive ligand-receptor assays has been this inverse relationship between intensity of the developed signal and sample ligand concentration. This relationship provides that a sample with a low concentration of ligand will produce a large signal in the assay; and conversely a sample with a high concentration of ligand will produce a small signal in the assay. A further disadvantage of such assays is that if the requirement is for a single test to simultaneously determine multiple ligands each of which must be assigned a semi-quantitative value and each of which has specific individual concentration targets, then individual specific reference zones would have to be provided for each ligand to be determined. Under such circumstances, a test for multiple ligands becomes difficult to produce and complex to interpret.
Methods have been described in European Patent Applications 87309723.2 and 87309724.0 and in PCT App. No. PCT/US86/00668 (International Publication Number WO 86/06170) where a signal is not developed in the assay until the ligand in the sample exceeds a predetermined amount. These methods utilize ligand receptors immobilized on a solid phase in an array that permits the contact of the ligand in the sample and the ligand analogue conjugate with the immobilized receptors. The contact is made in a chromatographic manner so that the liquid containing the ligand is drawn through the solid phase array in a directional manner. The binding capacity of the solid phase is empirically adjusted so that a predetermined amount of the ligand is bound by the solid phase during the transit of the fluid containing the ligand through the solid phase. The ligand, ligand analogue conjugate, and the array of immobilized receptor do not reach equilibrium during the assay process in these methods. Those skilled in the art will appreciate that the binding capacity of the immobilized receptor is highly dependent upon the immobilization conditions and their affect on the affinity of the receptor for the ligand and on the time during which the ligand and ligand analogue conjugate are able to bind to the immobilized receptor. The reliance of these methods on non-equilibrium conditions causes the manufacture of such assays to be unpredictable and difficult to reproduce. In the present invention equilibrium methods are utilized in order to be able to predict the behavior of assays so that the development of the assay is straightforward and the performance of the assay is reproducible.
The present invention is further directed to simplified methods for the assay of ligands that yield quantitative results. Those skilled in the art will appreciate that quantitative assays require calibration to achieve precise and accurate results. Because competitive assays generally result in non-linear response functions, several calibration points are required for such assays in order to determine the response over the assay range. In order to simplify the calibration process, two extreme approaches have evolved in the prior art. One approach is not to reduce the number of calibrators or replicates needed to determine the response but to reduce the frequency of such calibration. Such assays rely upon instruments to perform the assay and to control variables that affect the assay response so that calibration is infrequent or is performed by the manufacturer and does not need to be performed by the user of the assay. The second approach is to not use an instrument and to provide a simplified means of calibration so that no additional tests are needed to calibrate the assay response. The present invention provides novel methods of calibration which are simple to use in both instrument-based assays and assays that are visually interpreted.
The method of U.S. Pat. No. 4,540,659 provides an assay for the quantitation of ligand in samples where predetermined ratios of responses at a calibration surface and a measurement surface are related to the concentration of the ligand. While this method may provide a crude means of quantitation, it does not offer the precision or the accuracy of existing methods that utilize instruments nor does it provide quantitation without the use of instruments.
Another prior art approach, a non-competitive immunochromatographic assay, is described in U.S. Pat. Nos. 4,168,146 and 4,435,504. This assay provides a method for quantitatively determining the presence of a single analyte in a sample in a visually interpreted immunoassay but does not permit the assay of multiple analytes without employing multiple devices. Furthermore, in practice this method is restricted to ligands whose sample concentrations are high relative to ligands that are commonly determined by competitive assay technology. Accordingly, this type of approach is of limited utility. Clearly, there is an unmet need for a ligand-receptor assay capable of determining the presence of a multiplicity of ligands in a sample and concurrently providing individualized semi-quantitative results for each ligand. Furthermore, such an assay should produce such results in a format that is simple enough for an non-technical user to correctly perform and interpret. In addition there is a need for broadly applicable quantitative assay methods that are easily performed and interpreted. The inventive assays of this invention meet these requirements.
The present invention is a method for performing competitive ligand-receptor assays so as to be able semiquantitatively or quantitatively determine the concentration of the ligand. The invention permits the assay of the target ligand to be carried out such that the ligand concentration is determined relative to an internally specified concentration, the threshold concentration. The threshold concentration can be arbitrarily pre-selected to be equivalent to any concentration appropriate to the ligand of interest and serves as a calibration point for the assay of that ligand. The present invention provides quantitative methods that utilize the threshold concentration as a calibration point to enable simplified methods of quantitation. Furthermore, the present invention provides a method for performing competitive ligand-receptor assays for the simultaneous determination of a multiplicity of ligands, each determination including an internal threshold concentration specifically targeted for its respective ligand. One embodiment of the present invention is a method for performing competitive ligand-receptor assays for the simultaneous determination of a multiplicity of ligands, each determination including a compendium of internal threshold concentrations specifically targeted for the respective ligand. The method of the present invention affords the concentration determination to be carried out in a manner which is simple to employ and straightforward to interpret.